Elisa for vegf

ABSTRACT

The vascular endothelial growth factor (VEGF) activity in a patient&#39;s bloodstream or other biological sample can serve as a diagnostic and prognostic index for cancer, diabetes, heart conditions, and other pathologies. Antibody-sandwich ELISA methods and kits for VEGF as an antigen are provided to detect types of VEGF levels in biological samples from animal models and human patients and can be used as a diagnostic/prognostic index.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. Ser. No. 13/660,563, filed onOct. 25, 2012, now U.S. Pat. No. 9,146,245, which is a divisional ofU.S. Ser. No. 12/817,827, now U.S. Pat. No. 8,449,885, filed on Jun. 17,2010, which is a continuation of U.S. Ser. No. 11/866,560, filed on Oct.3, 2007, now abandoned, which claims priority to and the benefit of U.S.Provisional Application Ser. No. 60/828,203, filed on Oct. 4, 2006. Allof these applications are hereby incorporated by reference.

FIELD OF THE INVENTION

This invention relates to immunoassays for detecting certain populationsof VEGF that can be used as diagnostic and prognostic methods forpatients with cancer, cardiovascular, or other pathologies.

BACKGROUND

It is now well established that angiogenesis is implicated in thepathogenesis of a variety of disorders. These include solid tumors,intra-ocular neovascular syndromes such as proliferative retinopathiesor age-related macular degeneration (AMD), rheumatoid arthritis, andpsoriasis (Folkman et al. J. Biol. Chem. 267:10931-10934 (1992);Klagsbrun et al. Annu. Rev. Physiol. 53:217-239 (1991); and Garner A,Vascular diseases. In: Pathobiology of ocular disease. A dynamicapproach. Garner A, Klintworth G K, Eds. 2^(nd) Edition (Marcel Dekker,NY, 1994), pp 1625-1710). In the case of solid tumors, theneovascularization allows the tumor cells to acquire a growth advantageand proliferative autonomy compared to the normal cells. Accordingly, acorrelation has been observed between density of microvessels in tumorsections and patient survival in breast cancer as well as in severalother tumors (Weidner et al. N Engl J Med 324:1-6 (1991); Horak et al.Lancet 340:1120-1124 (1992); and Macchiarini et al. Lancet 340:145-146(1992)).

The search for positive regulators of angiogenesis has yielded manycandidates, including, e.g., aFGF, bFGF, TGF-α, TGF-β, HGF, TNF-α,angiogenin, IL-8, etc. (Folkman et al., supra, and Klagsbrun et al.,supra). Some of the negative regulators so far identified includethrombospondin (Good et al. Proc. Natl. Acad. Sci. USA. 87:6624-6628(1990)), the 16-kilodalton N-terminal fragment of prolactin (Clapp etal. Endocrinology, 133:1292-1299 (1993)), angiostatin (O'Reilly et al.Cell 79:315-328 (1994)), and endostatin (O'Reilly et al. Cell 88:277-285(1996)).

Work done over the last several years has established the key role ofvascular endothelial growth factor (VEGF) in the regulation of normaland abnormal angiogenesis (Ferrara et al. Endocr. Rev. 18:4-25 (1997)).The finding that the loss of even a single VEGF allele results inembryonic lethality points to an irreplaceable role played by thisfactor in the development and differentiation of the vascular system(Ferrara et al., supra).

Furthermore, VEGF has been shown to be a key mediator ofneovascularization associated with tumors and intra-ocular disorders(Ferrara et al., supra). The VEGF mRNA is overexpressed by the majorityof human tumors examined (Berkman et al. J Clin Invest 91:153-159(1993); Brown et al. Human Pathol. 26:86-91 (1995); Brown et al. CancerRes. 53:4727-4735 (1993); Mattern et al. Brit. J. Cancer. 73:931-934(1996); and Dvorak et al. Am J. Pathol. 146:1029-1039 (1995)). Also, theconcentration of VEGF in eye fluids is highly correlated to the presenceof active proliferation of blood vessels in patients with diabetic andother ischemia-related retinopathies (Aiello et al. N. Engl. J. Med.331:1480-1487 (1994)). Furthermore, studies have demonstrated thelocalization of VEGF in choroidal neovascular membranes in patientsaffected by acute macular degeneration (AMD) (Lopez et al. Invest.Ophtalmo. Vis. Sci. 37:855-868 (1996)).

VEGF is produced by tissues and does not have to enter the circulationto exert its biological effect, but rather acts locally as a paracrineregulator. A recent study by Yang et al. J. Pharm. Exp. Ther. 284:103(1998) found the clearance of rhVEGF₁₆₅ from the circulation to be veryrapid, suggesting endogenous VEGF in the circulation is most likely theresult of continual synthesis of VEGF. In addition, several studies havetried to correlate levels of circulating VEGF with tumor burden and havesuggested VEGF levels as a potential prognostic marker (Ferrari andScagliotti Eur. J. Cancer 32A:2368 (1996); Gasparini et al. J. Natl.Cancer Inst. 89:139 (1997); Kohn Cancer 80:2219 (1997); Baccala et al.Urology 51:327 (1998); Fujisaki et al. Am. J. Gastroenterol. 93:249(1998)). Clearly the ability to accurately measure VEGF will beimportant to understand its potential role(s) in many biologicalprocesses, such as maintenance of vascular patency, menstrual cycle,ischemia, diabetes, cancer, intraocular disorders, etc.

The literature reports widely varying concentrations of endogenous VEGFin normal and diseased patients, ranging from undetectable to highlevels. The ability to measure endogenous VEGF levels depends on theavailability of sensitive and specific assays. Colorimetric,chemiluminescence, and fluorometric based enzyme-linked immunosorbentassays (ELISAs) for VEGF have been reported. Houck et al., supra,(1992); Yeo et al. Clin. Chem. 38:71 (1992); Kondo et al. Biochim.Biophys. Acta 1221:211 (1994); Baker et al. Obstet. Gynecol. 86:815(1995); Hanatani et al. Biosci. Biotechnol. Biochem. 59:1958 (1995);Leith and Michelson Cell Prolif. 28:415 (1995); Shifren et al. J. Clin.Endocrinol. Metab. 81:3112 (1996); Takano et al. Cancer Res. 56:2185(1996); Toi et al. Cancer 77:1101 (1996); Brekken et al. Cancer Res.58:1952 (1998); Obermair et al. Br. J. Cancer 77:1870-1874 (1998); Webbet al. Clin. Sci. 94:395-404 (1998).

For example, Houck et al., supra (1992) describe a colorimetric ELISAthat appears to have ng/ml sensitivity, which may not be sensitiveenough to detect endogenous VEGF levels. Yeo et al., supra (1992)describe a two-site time-resolved immunofluorometric assay, however, noVEGF was detected in normal sera (Yeo et al. Cancer Res. 53:2912(1993)). Baker et al., supra (1995), using a modified version of thisimmunofluorometric assay, reported detectable levels of VEGF in plasmafrom pregnant women, with higher levels observed in women withpreeclampsia. Similar data in pregnant women were reported by Anthony etal. Ann. Clin. Biochem. 34:276 (1997) using a radioimmunoassay. Hanataniet al., supra (1995) developed a chemiluminescent ELISA capable ofmeasuring circulating VEGF and report VEGF levels in sera from 30 normalindividuals (male and female) from 8-36 pg/ml. Brekken et al, supra(1998) described ELISA assays using antibodies having binding preferenceto either the VEGF alone or the VEGF:Flk-1 complex.

An ELISA kit for VEGF detection is commercially available from R&DSystems (Minneapolis, Minn.). The R&D VEGF ELISA kit has been used insandwich assays wherein a monoclonal antibody is used to capture thetarget VEGF antigen and a polyclonal antibody is used to detect theVEGF. Webb et al. supra (1998). See, also, e.g., Obermair et al., supra(1998).

Keyt et al. J. Biol. Chem. 271:7788-7795 (1996); Keyt et al. J. Biol.Chem. 271:5638 (1996); and Shifren et al., supra (1996) also developed acolorimetric ELISA based on a dual monoclonal antibody pair. Althoughthis ELISA was able to detect elevated VEGF levels in cancer patients,it lacked the sensitivity needed to measure endogenous levels of VEGF innormal individuals. Rodriguez et al. J. Immunol. Methods 219:45 (1998)described a two-site fluorimetric VEGF ELISA that yields a sensitivityof 10 pg/ml VEGF in neat plasma or serum. However, this fluorimetricassay detects fully intact 165/165 and 165/110 species of VEGF (It hasbeen reported that VEGF 165/165 can be proteolytically cleaved intothree other forms: a 165/110 heterodimer, a 110/110 homodimer, and a55-amino-acid C-terminal fragment (Keyt et al. J. Biol. Chem.271:7788-7795 (1996); Keck et al. Arch. Biochem. Biophys. 344:103-113(1997)).).

Thus, there is a need to develop a diagnostic and prognostic assay thatdetects higher measurable levels of VEGF in a biological sample of ananimal model or patient than existing ELISAs, and/or can measuredifferent isoforms of VEGF.

SUMMARY

Antibody-sandwich ELISA methods for VEGF as an antigen were developed todetect VEGF forms in biological samples. The VEGF ELISA provided hereinis capable of detecting VEGF isoforms and fragments of VEGF greater than110 (“VEGF₁₁₀₊”). Kits thereof are also provided.

For example, methods for detecting selective vascular endothelial growthfactor (VEGF) forms greater than 110 amino acids (VEGF₁₁₀₊) in abiological sample comprise the steps of: (a) contacting and incubatingthe biological sample with a capture reagent immobilized to a solidsupport, wherein the capture reagent is an antibody that recognizes sameepitope as antibody 5C3 against human VEGF, said monoclonal antibodybinding specifically to residues greater than 110 of human VEGF; (b)separating the biological sample from the immobilized capture reagents;(c) contacting the immobilized capture reagent-target molecule complexwith a detectable antibody that binds to the KDR and/or FLT1 receptorbinding domains of VEGF; and (d) measuring the level of VEGF₁₁₀₊ boundto the capture reagents using a detection means for the detectableantibody. In certain embodiments, the detectable antibody binds to anepitope in VEGF 1-110. In certain embodiments, comparison ELISA can beperformed to detect different types of VEGF. In certain embodiments, thebiological sample (e.g., tumor samples or tumor lysates, plasma, serum,or urine, etc.) is isolated from a human subject.

In one embodiment, the capture reagent is the 5C3 monoclonal antibody.In one embodiment, the immobilized capture reagent is coated on amicrotiter plate. In certain embodiments, the detectable antibody is amonoclonal antibody. In one embodiment, the detectable antibody is amurine monoclonal antibody. In one embodiment, the immobilizedmonoclonal antibody is MAb 5C3 and the detectable antibody is MAbA4.6.1. In certain embodiments, the detectable antibody is directlydetectable. In one embodiment, detectable antibody is amplified by acolorimetric reagent. In one embodiment, the detectable antibody isbiotinylated and the detection means is avidin orstreptavidin-peroxidase and 3,3′,5,5′-tetramethyl benzidine.

In certain embodiments of the invention, the human subject is avascular, diabetic, or cancer patient and the measuring step (d) furthercomprises a comparison with a standard curve to determine the level ofVEGF compared to a normal individual.

Kits are also provided. For example, an immunoassay kit for detectingvascular endothelial growth factor (VEGF) forms greater than 110 aminoacids (VEGF₁₁₀₊) in a biological sample can comprise: (a) as capturereagent, an antibody against human VEGF, wherein the monoclonal antibodybinds specifically to the residues greater than 110 of human VEGF; and(b) as detection reagent, a detectable antibody that binds to the KDRand/or FLT1 receptor binding domains of VEGF. In certain embodiments,the detectable antibody binds to an epitope in VEGF 1-110. In certainembodiments, the kit, further comprises a solid support for the capturereagents. For example, the capture reagents can be immobilized on thesolid support (e.g., a microtiter plate). In certain embodiments, thekit further comprises a detection means (e.g., colormetric means,fluorimetric means, etc.) for the detectable antibodies. In certainembodiments, the kit further comprises purified VEGF as an antigenstandard. In certain embodiments of the invention, an additional VEGFELISA or more can be provided for comparison studies with the VEGF₁₁₀₊ELISA. In one embodiment, the kit includes a capture reagent monoclonalantibody, which is murine monoclonal antibody MAb 5C3, and a detectableantibody, which is MAb A4.6.1.

In yet another embodiment, the invention provides an anti-VEGF antibody5C3 (obtainable from or produced by hybridoma deposited under ATCCnumber PTA-7737). The invention also provides an antibody that does notbind VEGF 1-110 and binds to the same VEGF₁₁₀₊ epitope as the monoclonalantibody produced by hybridoma cell line PTA-7737. In certainembodiments, an antibody of the invention is conjugated to a detectablelabel. In one embodiment, the hybridoma 5C3.1.1 deposited under ATCCdeposit number PTA-7737 is provided.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A-1C illustrate the detection of recombinant VEGF165, VEGF121(1)(truncated, likely missing approximately 9 amino acids from thecarboxy-terminus according to the manufacturer, R&D systems), VEGF121(2)(from Pepro Tech), VEGF110 (N-terminal fragment generated by plasmindigestion of VEGF) and VEGF8-109 (artificial VEGF with amino acids 8-109of VEGF165) molecules by different VEGF ELISAs. FIG. 1A: ELISA A using3.5F8 for coat and biotinylated A4.6.1 for detection. FIG. 1B: ELISA Busing A4.6.1 for coat and biotinylate 2E3 for detection. FIG. 1C: ELISAC using 5C3 for coat and biotinylated A4.6.1 for detection.

FIG. 2 illustrates protein blotting of VEGF produced by A673 cells,using 3.5F8 (left) or A4.6.1 (right) for probing. Samples are VEGFpurified from conditioned medium of A673 cells using an A4.6.1 Affinitycolumn (lane 1) and recombinant VEGF proteins VEGF₁₆₅, VEGF₁₂₁ (likelymissing approximately 9 amino acids from the carboxy-terminus accordingto the manufacturer, R&D systems) and VEGF₈₋₁₀₉ produced by E.coli(lanes 2, 3 and 4, respectively).

FIG. 3 illustrates a diagram of VEGF₁₆₅, VEGF₁₂₁ and VEGF₁₁₀ (N-terminalfragment generated by plasmin digestion of VEGF) showing the proposedbinding sites of the antibodies used in the three VEGF ELISAs.

DETAILED DESCRIPTION Definitions

Before describing the present invention in detail, it is to beunderstood that this invention is not limited to particular compositionsor biological systems, which can, of course, vary. It is also to beunderstood that the terminology used herein is for the purpose ofdescribing particular embodiments only, and is not intended to belimiting. As used in this specification and the appended claims, thesingular forms “a”, “an” and “the” include plural referents unless thecontent clearly dictates otherwise. Thus, for example, reference to “amolecule” optionally includes a combination of two or more suchmolecules, and the like.

The term “VEGF” as used herein refers to the 165-amino acid vascularendothelial cell growth factor, and related 121-, 145-, 189-, and206-amino acid vascular endothelial cell growth factors, as described byLeung et al. Science 246:1306 (1989), Houck et al. Mol. Endocrin. 5:1806(1991), and Neufeld et al., supra, together with the naturally occurringallelic and processed forms of those growth factors. See also, e.g.,FIGS. 1A and B of U.S. Pat. No. 6,057,428. Active VEGF fragment can bereleased from ECM-bound VEGF by plasmin cleavage, generating the first110 amino acids (see, e.g., Keyt B A, et al.,: The carboxyl-terminaldomain (111-165) of vascular endothelial growth factor is critical forits mitogenic potency. J Biol Chem. 271: 7788-7795 (1996)). “VEGF₁₁₀₊”as used herein refers to VEGF fragments that are greater than 110 aminoacids (from the N-terminal), but do not include the first 110 aminoacids or smaller fragments (e.g., VEGF₈₋₁₀₉).

The term “detecting” is used in the broadest sense to include bothqualitative and quantitative measurements of a target molecule. In oneaspect, the detecting method as described herein is used to identify themere presence of VEGF₁₁₀₊ or VEGF in a biological sample. In anotheraspect, the method is used to test whether VEGF₁₁₀₊ or VEGF in a sampleis at a detectable level. In yet another aspect, the method can be usedto quantify the amount of VEGF₁₁₀₊ or VEGF in a sample and further tocompare the VEGF₁₁₀₊ or VEGF levels from different samples.

The term “biological sample” refers to a body sample from any animal,but preferably is from a mammal, more preferably from a human. Incertain embodiments, such biological sample is from a vascular,diabetic, or cancer patient. Such samples include biological fluids suchas serum, plasma, vitreous fluid, lymph fluid, synovial fluid,follicular fluid, seminal fluid, amniotic fluid, milk, whole blood,urine, cerebro-spinal fluid, saliva, sputum, tears, perspiration, mucus,tumor lysates, and tissue culture medium, as well as tissue extractssuch as homogenized tissue, tumor tissue, and cellular extracts. Incertain embodiments, the sample is a body sample from any animal, in oneembodiment it is from a mammal, in one embodiment from a human subject.In one embodiment, such biological sample is from clinical patients.

The term “detectable antibody” refers to an antibody that is capable ofbeing detected either directly through a label amplified by a detectionmeans, or indirectly through, e.g., another antibody that is labeled.For direct labeling, the antibody is typically conjugated to a moietythat is detectable by some means. In one embodiment, the detectableantibody is biotinylated antibody.

The term “detection means” refers to a moiety or technique used todetect the presence of the detectable antibody in the ELISA herein andincludes detection agents that amplify the immobilized label such aslabel captured onto a microtiter plate. In one embodiment, the detectionmeans is a colorimetric detection agent such as avidin orstreptavidin-HRP.

The term “capture reagent” refers to a reagent capable of binding andcapturing a target molecule in a sample such that under suitablecondition, the capture reagent-target molecule complex can be separatedfrom the rest of the sample. Typically, the capture reagent isimmobilized or immobilizable. In a sandwich immunoassay, the capturereagent is preferably an antibody or a mixture of different antibodiesagainst a target antigen.

The term “antibody” herein is used in the broadest sense andspecifically covers intact monoclonal antibodies, polyclonal antibodies,multispecific antibodies (e.g. bispecific antibodies) formed from atleast two intact antibodies, and antibody fragments so long as theyexhibit the desired biological activity.

“Antibody fragments” comprise a portion of an intact antibody,preferably comprising the antigen-binding or variable region thereof.Examples of antibody fragments include Fab, Fab′, F(ab′)₂, and Fvfragments; diabodies; linear antibodies; single-chain antibodymolecules; and multispecific antibodies formed from antibody fragments.

For the purposes herein, an “intact antibody” is one comprising heavy-and light-chain variable domains as well as an Fc region.

“Native antibodies” are usually heterotetrameric glycoproteins of about150,000 daltons, composed of two identical light (L) chains and twoidentical heavy (H) chains. Each light chain is linked to a heavy chainby one covalent disulfide bond, while the number of disulfide linkagesvaries among the heavy chains of different immunoglobulin isotypes. Eachheavy and light chain also has regularly spaced intrachain disulfidebridges. Each heavy chain has at one end a variable domain (V_(H))followed by a number of constant domains. Each light chain has avariable domain at one end (V_(L)) and a constant domain at its otherend; the constant domain of the light chain is aligned with the firstconstant domain of the heavy chain, and the light-chain variable domainis aligned with the variable domain of the heavy chain. Particular aminoacid residues are believed to form an interface between the light-chainand heavy-chain variable domains.

The term “monoclonal antibody” as used herein refers to an antibodyobtained from a population of substantially homogeneous antibodies,i.e., the individual antibodies comprising the population are identicalexcept for possible naturally occurring mutations that may be present inminor amounts. Monoclonal antibodies are highly specific, being directedagainst a single antigenic site. Furthermore, in contrast toconventional (polyclonal) antibody preparations that typically includedifferent antibodies directed against different determinants (epitopes),each monoclonal antibody is directed against a single determinant on theantigen. In addition to their specificity, the monoclonal antibodies areadvantageous in that they are synthesized by the hybridoma culture,uncontaminated by other immunoglobulins. The modifier “monoclonal”indicates the character of the antibody as being obtained from asubstantially homogeneous population of antibodies, and is not to beconstrued as requiring production of the antibody by any particularmethod. For example, the monoclonal antibodies to be used in accordancewith the present invention may be made by the hybridoma method firstdescribed by Kohler et al., Nature, 256:495 (1975), or may be made byrecombinant DNA methods (see, e.g., U.S. Pat. No. 4,816,567). The“monoclonal antibodies” may also be isolated from phage antibodylibraries using the techniques described in Clackson et al. Nature352:624-628 (1991) and Marks et al., J. Mol. Biol., 222:581-597 (1991),for example.

The monoclonal antibodies herein specifically include “chimeric”antibodies (immunoglobulins) in which a portion of the heavy and/orlight chain is identical with or homologous to corresponding sequencesin antibodies derived from a particular species or belonging to aparticular antibody class or subclass, while the remainder of thechain(s) is identical with or homologous to corresponding sequences inantibodies derived from another species or belonging to another antibodyclass or subclass, as well as fragments of such antibodies, so long asthey exhibit the desired biological activity (U.S. Pat. No. 4,816,567;Morrison et al., Proc. Natl. Acad. Sci. USA, 81:6851-6855 (1984)).Chimeric antibodies of interest herein include “primatized” antibodiescomprising variable-domain antigen-binding sequences derived from anon-human primate (e.g. Old World Monkey, such as baboon, rhesus orcynomolgus monkey) and human constant-region sequences (U.S. Pat. No.5,693,780).

“Humanized” forms of non-human (e.g., murine) antibodies are chimericantibodies that contain minimal sequence derived from non-humanimmunoglobulin. For the most part, humanized antibodies are humanimmunoglobulins (recipient antibody) in which residues from ahypervariable region of the recipient are replaced by residues from ahypervariable region of a non-human species (donor antibody) such asmouse, rat, rabbit or non-human primate having the desired specificity,affinity, and capacity. In some instances, framework region (FR)residues of the human immunoglobulin are replaced by correspondingnon-human residues. Furthermore, humanized antibodies may compriseresidues that are not found in the recipient antibody or in the donorantibody. These modifications are made further to refine antibodyperformance. In general, the humanized antibody will comprisesubstantially all of at least one, and typically two, variable domains,in which all or substantially all of the hypervariable loops correspondto those of a non-human immunoglobulin and all or substantially all ofthe FRs are those of a human immunoglobulin sequence. The humanizedantibody optionally also will comprise at least a portion of animmunoglobulin constant region (Fc), typically that of a humanimmunoglobulin. For further details, see Jones et al., Nature,321:522-525 (1986); Riechmann et al., Nature, 332:323-329 (1988); andPresta, Curr. Op. Struct. Biol., 2:593-596 (1992). In one embodiment, ahumanized 5C3 antibody is provided and used the methods provided herein.

The term “variable” refers to the fact that certain portions of thevariable domains differ extensively in sequence among antibodies and areused in the binding and specificity of each particular antibody for itsparticular antigen. However, the variability is not evenly distributedthroughout the variable domains of antibodies. It is concentrated inthree segments called hypervariable regions in both the light-chain andthe heavy-chain variable domains. The more highly conserved portions ofvariable domains are called the framework regions (FRs). The variabledomains of native heavy and light chains each comprise four FRs, largelyadopting a β-sheet configuration, connected by three hypervariableregions, which form loops connecting, and in some cases forming part of,the β-sheet structure. The hypervariable regions in each chain are heldtogether in close proximity by the FRs and, with the hypervariableregions from the other chain, contribute to the formation of theantigen-binding site of antibodies (see Kabat et al., Sequences ofProteins of Immunological Interest, 5^(th) Ed. Public Health Service,National Institutes of Health, Bethesda, Md. (1991)). The constantdomains are not involved directly in binding an antibody to an antigen,but exhibit various effector functions, such as participation of theantibody in antibody-dependent cellular cytotoxicity (ADCC).

Papain digestion of antibodies produces two identical antigen-bindingfragments, called “Fab” fragments, each with a single antigen-bindingsite, and a residual “Fc” fragment, whose name reflects its ability tocrystallize readily. Pepsin treatment yields an F(ab′)₂ fragment thathas two antigen-binding sites and is still capable of cross-linkingantigen.

“Fv” is the minimum antibody fragment that contains a completeantigen-recognition and antigen-binding site. This region consists of adimer of one heavy-chain and one light-chain variable domain in tight,non-covalent association. It is in this configuration that the threehypervariable regions of each variable domain interact to define anantigen-binding site on the surface of the V_(H)-V_(L) dimer.Collectively, the six hypervariable regions confer antigen-bindingspecificity to the antibody. However, even a single variable domain (orhalf of an Fv comprising only three hypervariable regions specific foran antigen) has the ability to recognize and bind antigen, although at alower affinity than the entire binding site.

The Fab fragment also contains the constant domain of the light chainand the first constant domain (CH1) of the heavy chain. Fab′ fragmentsdiffer from Fab fragments by the addition of a few residues at thecarboxy terminus of the heavy-chain CH1 domain including one or morecysteines from the antibody hinge region. Fab′-SH is the designationherein for Fab′ in which the cysteine residue(s) of the constant domainsbear at least one free thiol group. F(ab′)₂ antibody fragmentsoriginally were produced as pairs of Fab′ fragments that have hingecysteines between them. Other chemical couplings of antibody fragmentsare also known.

The “light chains” of antibodies (immunoglobulins) from any vertebratespecies can be assigned to one of two clearly distinct types, calledkappa (κ) and lambda (λ), based on the amino acid sequences of theirconstant domains.

Depending on the amino acid sequence of the constant domain of theirheavy chains, antibodies can be assigned to different classes. There arefive major classes of intact antibodies: IgA, IgD, IgE, IgG, and IgM,and several of these may be further divided into subclasses (isotypes),e.g., IgG1, IgG2, IgG3, IgG4, IgA, and IgA2. The heavy-chain constantdomains that correspond to the different classes of antibodies arecalled α, δ, ε, γ and μ, respectively. The subunit structures andthree-dimensional configurations of different classes of immunoglobulinsare well known.

“Single-chain Fv” or “scFv” antibody fragments comprise the V_(H) andV_(L) domains of antibody, wherein these domains are present in a singlepolypeptide chain. Preferably, the Fv polypeptide further comprises apolypeptide linker between the V_(H) and V_(L) domains that enables thescFv to form the desired structure for antigen binding. For a review ofscFv, see Pluckthun in The Pharmacology of Monoclonal Antibodies, vol.113, Rosenburg and Moore eds., Springer-Verlag, New York, pp. 269-315(1994).

The term “hypervariable region” when used herein refers to the aminoacid residues of an antibody that are responsible for antigen binding.The hypervariable region comprises amino acid residues from a“complementarity-determining region” or “CDR” (e.g. residues 24-34 (L1),50-56 (L2) and 89-97 (L3) in the light-chain variable domain and 31-35(H1), 50-65 (H2) and 95-102 (H3) in the heavy-chain variable domain;Kabat et al., Sequences of Proteins of Immunological Interest, 5^(th)Ed. Public Health Service, National Institutes of Health, Bethesda, Md.(1991)) and/or those residues from a “hypervariable loop” (e.g. residues26-32 (L1), 50-52 (L2) and 91-96 (L3) in the light-chain variable domainand 26-32 (H1), 53-55 (H2) and 96-101 (H3) in the heavy-chain variabledomain; Chothia and Lesk J. Mol. Biol. 196:901-917 (1987)). “Framework”or “FR” residues are those variable domain residues other than thehypervariable region residues as herein defined.

“Mammal” for purposes of treatment refers to any animal classified as amammal, including humans, domestic, and farm animals, and zoo, sports,or pet animals, such as dogs, horses, cats, sheep, pigs, cows, etc.Preferably, the mammal is human.

The terms “cancer”, “cancerous”, and “malignant” refer to or describethe physiological condition in mammals that is typically characterizedby unregulated cell growth. Examples of cancer include but are notlimited to, carcinoma including adenocarcinoma, lymphoma, blastoma,melanoma, sarcoma, and leukemia. More particular examples of suchcancers include squamous cell cancer, lung cancer (including small-celllung cancer, non-small cell lung cancer, adenocarcinoma of the lung, andsquamous carcinoma of the lung), cancer of the peritoneum,hepatocellular cancer, gastric or stomach cancer (includinggastrointestinal cancer), gastrointestinal stromal cancer, pancreaticcancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer(e.g., hepatic carcinoma and hepatoma), bladder cancer, hepatoma, breastcancer, colon cancer, colorectal cancer, rectal cancer, endometrial oruterine carcinoma, salivary gland carcinoma, kidney or renal cancer,liver cancer, prostate cancer, vulval cancer, thyroid cancer, basal cellcarcinoma, testicular cancer, esophageal cancer, hepatic carcinoma,soft-tissue sarcoma, kaposi's sarcoma, carcinoid carcinoma,mesothelioma, multiple myeloma, and various types of head and neckcancer, as well as B-cell lymphoma (including low grade/follicularnon-Hodgkin's lymphoma (NHL); small lymphocytic (SL) NHL; intermediategrade/follicular NHL; intermediate grade diffuse NHL; high gradeimmunoblastic NHL; high grade lymphoblastic NHL; high grade smallnon-cleaved cell NHL; bulky disease NHL; mantle cell lymphoma;AIDS-related lymphoma; and Waldenstrom's Macroglobulinemia); Hodgkin'slymphoma, chronic lymphocytic leukemia (CLL), acute lymphoblasticleukemia (ALL), Hairy cell leukemia; chronic myeloblastic leukemia, andpost-transplant lymphoproliferative disorder (PTLD), as well as abnormalvascular proliferation associated with phakomatoses, edema (such as thatassociated with brain tumors), and Meigs' syndrome.

The phrases “vascular” and “cardiovascular” are used interchangeably anddescribe patients with indications that stimulate angiogenesis and/orcardiovascularization, and those that inhibit angiogenesis and/orcardiovascularization. Such disorders include, for example, arterialdisease, such as atherosclerosis, hypertension, inflammatory vasculitis,Reynaud's disease and Reynaud's phenomenon, aneurysms, and arterialrestenosis; venous and lymphatic disorders such as thrombophlebitis,lymphangitis, and lymphedema; and other vascular disorders such asperipheral vascular disease, AMD, cancer such as vascular tumors, e.g.,hemangioma (capillary and cavernous), glomus tumors, telangiectasia,bacillary angiomatosis, hemangioendothelioma, angiosarcoma,haemangiopericytoma, Kaposi's sarcoma, lymphangioma, andlymphangiosarcoma, tumor angiogenesis, trauma such as wounds, burns, andother injured tissue, implant fixation, scarring, ischemia reperfusioninjury, rheumatoid arthritis, cerebrovascular disease, renal diseasessuch as acute renal failure, and osteoporosis. This would also includeangina, myocardial infarctions such as acute myocardial infarctions,cardiac hypertrophy, and heart failure such as congestive heart failure(CHF).

The term “diabetes” refers to a progressive disease of carbohydratemetabolism involving inadequate production or utilization of insulin andis characterized by hyperglycemia and glycosuria. This term includes allforms of diabetes, such as type I and type II diabetes andinsulin-resistant diabetes, such as Mendenhall's Syndrome, WernerSyndrome, leprechaunism, lipoatrophic diabetes, and other lipoatrophies.

The term “affinity purified” refers to purifying a substance by elutingit through an affinity chromatography column.

ELISA

Vascular endothelial growth factor (VEGF) is a homodimeric glycoproteinand is a key angiogenic factor of blood vessel formation duringdevelopment and in pathological angiogenesis associated with tumors.Expression of VEGF is potentiated in response to hypoxia, andpotentially other factors such as growth factors, hormones andoncogenes. (see, e.g., Ferrara N: Vascular endothelial growth factor:Basic science and clinical progress. Endocrine Reviews 25: 581-611(2004)). The human VEGF gene has eight exons separated by introns.Alternative RNA splicing results in the generation of at least fourmajor isoforms having 121, 165, 189 and 206 amino acids in monomer (see,e.g., Houck K A, et al.,: The vascular endothelial growth factor family:identification of a fourth molecular species and characterization ofalternative splicing of RNA. Mol Endocrinol 5: 1806-1814 (1991); and,Tischer E, et al.,: The human gene for vascular endothelial growthfactor. Multiple protein forms are encoded through alternative exonsplicing. J Biol Chem 266: 11947-11954 (1991)). Less frequent isoformsincluding those having 145 (see, e.g., Poltorak Z., et al.,: VEGF145, asecreted vascular endothelial growth factor isoform that binds toextracellular matrix. J Biol Chem 272: 7151-7158 (1997)) and 183 (see,e.g., Jingjing L, et al.,: Human Muller cells express VEGF183, a novelspliced variant of vascular endothelial growth factor. Invest OphthalmolVis Sci 40:752-759 (1999)) amino acids in monomer have also beenreported. All VEGF isoforms bind two receptor tyrosine kinases, VEGFR-1(see, e.g., De Vries C, et al.,: The fms-like tyrosine kinase, areceptor for vascular endothelial growth factor. Science 255:989-991(1992)) and VEGFR-2 (see, e.g., Terman B I, et al.,: Identification of anew endothelial cell growth factor receptor tyrosine kinase. Oncogene6:1677-1683 (1991)). VEGF₁₆₅ also interacts with neuropilin (see, e.g.,Soker S. et al.,: Neuropilin-1 is expressed by endothelial and tumorcells as an isoform-specific receptor for vascular endothelial growthfactor. Cell 92: 735-745 (1998)). VEGF₁₈₉ and VEGF₂₀₆ bind to heparinwith a high affinity and are mostly sequestered in the extracellularmatrix (ECM). VEGF₁₆₅ binds to heparin with intermediate affinity and ispartially soluble and partially bound to cell surface and ECM. VEGF₁₂₁does not bind to heparin and is freely soluble. VEGF₁₂₁ and VEGF₁₆₅ werefound to be the most dominantly expressed variants in breast and ovariancancer tumor specimens and cell lines by reverse transcription—PCRanalysis, while VEGF₂₀₆ expression was not detected. VEGF₁₈₃ and VEG₁₈₉expression was found to be non-detectable or at low levels in the celllines and were detected in some of the tumor specimens (see, e.g.,Stimpfl M, et al.,: Vascular Endothelial growth factor splice variantsand their prognostic value in breast and ovarian cancer. Clinical CancerResearch 8: 2253-2259 (2002)).

Active VEGF fragment can be released from ECM-bound VEGF by plasmincleavage, generating the first 110 amino acids (see, e.g., Keyt B A, etal., The carboxyl-terminal domain (111-165) of vascular endothelialgrowth factor is critical for its mitogenic potency. J Biol Chem. 271:7788-7795 (1996)). This could be a mechanism to locally regulate thebioavailability of VEGF during physiological and pathological processesof angiogenesis. See, e.g., Houck K A, et al. Dual regulation ofvascular endothelial growth factor bioavailability by genetic andproteolytic mechanisms. J Biol Chem 1992; 267:26031-26037 (1992); Keyt BA, et al. The carboxy-terminal domain (111-165) of vascular endothelialgrowth factor is critical for its mitogenic potency. J Biol Chem.271:7788-7795 (1996); and, Roth D, et al. Plasmin modulates vascularendothelial growth factor-A-mediated angiogenesis during wound repair.Am Pathology 168: 670-684. (10-12) (2006). However, VEGF₁₁₀concentrations in biological samples have not been reported. Active VEGFfragments may also be released from ECM-bound VEGF by matrixmetalloproteinase (MMP) cleavage. This is supported by the finding ofdegraded VEGF fragments with amino acids additional to 1-110 in ascitesfrom ovarian cancer patients. Both plasmin and MMP3 were detected in theascites. See, e.g., Lee S, Shahla M J, et al. Processing of VEGF-A bymatrix metalloproteinases regulates bioavailability and vascularpatterning in tumors. J Cell Biology 169:681-691 (2005).

Enzyme-linked immunosorbent assays (ELISAs) for various antigens includethose based on colorimetry, chemiluminescence, and fluorometry. ELISAshave been successfully applied in the determination of low amounts ofdrugs and other antigenic components in plasma and urine samples,involve no extraction steps, and are simple to carry out. The assaydescribed herein is an ELISA that utilizes antibodies as capturereagents and detectable antibodies for VEGF and VEGF₁₁₀₊. In certainembodiments, the ELISA is cell-based. In the first step of the assay thebiological sample suspected of containing VEGF or containing VEGF₁₁₀₊ iscontacted and incubated with the capture (or coat) antibodies so thatthe capture antibodies capture or bind to the VEGF or VEGF₁₁₀₊ so thatit can be detected in a detection step. The detection step involves useof the detectable antibody, which, when contacted with any of the boundVEGF or VEGF₁₁₀₊, binds to the protein of interest, if present, and adetection means is used to detect the label on the antibody and hencethe presence or amount of VEGF or VEGF₁₁₀₊ present. This ELISA can becompared with an ELISA that recognizes total VEGF (e.g., U.S. Pat. No.6,855,508; those described herein, and those known in the art) orisoforms of VEGF to determine the type of VEGF present.

For example, in certain embodiments, the assay utilizes the followingsteps.

First Step

In the first step of the assay herein, the biological sample iscontacted and incubated with the immobilized capture (or coat) reagent,which is an anti-VEGF monoclonal antibody. This antibody may be from anyspecies, but preferably the monoclonal antibody is a murine or ratmonoclonal antibody, more preferably murine, and most preferably MAb 5C3derived from the hybridoma identified herein. Hence, in a specificpreferred embodiment, the immobilized monoclonal antibody is a murinemonoclonal antibody, most preferably MAb 5C3. Immobilizationconventionally is accomplished by insolubilizing the capture reagenteither before the assay procedure, as by adsorption to a water-insolublematrix or surface (U.S. Pat. No. 3,720,760) or non-covalent or covalentcoupling (for example, using glutaraldehyde or carbodiimidecross-linking, with or without prior activation of the support with,e.g., nitric acid and a reducing agent as described in U.S. Pat. No.3,645,852 or in Rotmans et al. J. Immunol. Methods 57:87-98 (1983)), orafterward, e.g., by immunoprecipitation.

The solid phase used for immobilization may be any inert support orcarrier that is essentially water insoluble and useful in immunometricassays, including supports in the form of, e.g., surfaces, particles,porous matrices, etc. Examples of commonly used supports include smallsheets, Sephadex, polyvinyl chloride, plastic beads, and assay plates ortest tubes manufactured from polyethylene, polypropylene, polystyrene,and the like including 96-well microtiter plates, as well as particulatematerials such as filter paper, agarose, cross-linked dextran, and otherpolysaccharides. Alternatively, reactive water-insoluble matrices suchas cyanogen bromide-activated carbohydrates and the reactive substratesdescribed in U.S. Pat. Nos. 3,969,287; 3,691,016; 4,195,128; 4,247,642;4,229,537; and 4,330,440 are suitably employed for capture reagentimmobilization. In one embodiment the immobilized capture reagent iscoated on a microtiter plate, and in particular the preferred solidphase used is a multi-well microtiter plate that can be used to analyzeseveral samples at one time, e.g., a microtest 96-well ELISA plate suchas that sold as Nune Maxisorb or Immulon. In certain embodiments, theplate is a MICROTEST™ or MAXISORP™ 96-well ELISA plate such as that soldas NUNC MAXISORB™ or IMMULON™.

The solid phase is coated with the capture reagent as defined above,which may be linked by a non-covalent or covalent interaction orphysical linkage as desired. Techniques for attachment include thosedescribed in U.S. Pat. No. 4,376,110 and the references cited therein.If covalent, the plate or other solid phase is incubated with across-linking agent together with the capture reagent under conditionswell known in the art, e.g., such as for 1 hour at room temperature.

Commonly used cross-linking agents for attaching the capture reagent tothe solid phase substrate include, e.g.,1,1-bis(diazoacetyl)-2-phenylethane, glutaraldehyde,N-hydroxy-succinimide esters, for example, esters with 4-azido-salicylicacid, homobifunctional imidoesters, including disuccinimidyl esters suchas 3,3′-dithiobis-(succinimidyl-propionate), and bifunctional maleimidessuch as bis-N-maleimido-1,8-octane. Derivatizing agents such asmethyl-3-[(p-azidophenyl)-dithio]pro-pioimi-date yield photoactivatableintermediates capable of forming cross-links in the presence of light.

If 96-well plates are utilized, they are typically coated with thecapture reagent (typically diluted in a buffer such as 0.05 M sodiumcarbonate by incubation for at least about 10 hours, more preferably atleast overnight, at temperatures of about 4-20° C., or about 4-8° C.,and at a pH of about 8-12, or about pH 9-10, or about pH 9.6). Ifshorter coating times are desired, one can coat, e.g., 96-well plates atroom temperature for two hours. The plates may be stacked and coatedlong in advance of the assay itself, and then the assay can be carriedout simultaneously on several samples in a manual, semi-automatic, orautomatic fashion, such as by using robotics.

The coated plates are then typically treated with a blocking agent thatbinds non-specifically to and saturates the binding sites to preventunwanted binding of the free ligand to the excess sites on the wells ofthe plate. Examples of appropriate blocking agents for this purposeinclude, e.g., gelatin, bovine serum albumin, egg albumin, casein, andnon-fat milk. The blocking treatment typically takes place underconditions of ambient temperatures for about 1-4 hours, preferably about1 to 3 hours, or overnight at 0-4° C.

After coating and blocking, the VEGF standard (purified VEGF) or thebiological sample to be analyzed, appropriately diluted, is added to theimmobilized phase. The preferred dilution rate is about 1-15%,preferably about 10%, by volume. Buffers that may be used for dilutionfor this purpose include (a) PBS containing 0.5% BSA, 0.05% TWEEN 20™detergent (P20), 0.05% PROCLIN™ 300 antibiotic, 5 mM EDTA, 0.25% Chapssurfactant, 0.2% beta-gamma globulin, and 0.35M NaCl, pH 7.4; (b) PBScontaining 0.5% bovine serum albumin, 0.05% polysorbate 20, 5 mM EDTA,0.25% CHAPS, 0.2% bovine γ-globulins, and 0.35 M NaCl; pH 7.4 (c) PBScontaining 0.5% BSA, 0.05% polysorbate 20 (P20), and 0.05% PROCLIN™ 300,pH 7; (d) PBS containing 0.5% BSA, 0.05% P20, 0.05% PROCLIN™ 300, 5 mMEDTA, and 0.35 M NaCl, pH 6.35; (e) PBS containing 0.5% BSA, 0.05% P20,0.05% PROCLIN™ 300, 5 mM EDTA, 0.2% beta-gamma globulin, and 0.35 MNaCl, pH 7.4; and (f) PBS containing 0.5% BSA, 0.05% P20, 0.05% PROCLIN™300, 5 mM EDTA, 0.25% Chaps, and 0.35 M NaCl, pH 7.4. PROCLIN™ 300 actsas a preservative, and TWEEN 20™ acts as a detergent to eliminatenon-specific binding.

While the concentration of the capture reagents will generally bedetermined by the concentration range of interest of the VEGF taking anynecessary dilution of the biological sample into account, the finalconcentration of the capture reagent will normally be determinedempirically to maximize the sensitivity of the assay over the range ofinterest.

The conditions for incubation of sample and immobilized capture reagentare selected to maximize sensitivity of the assay and to minimizedissociation. Preferably, the incubation is accomplished at fairlyconstant temperatures, ranging from about 0° C. to about 40° C.,preferably from about 20 to 25° C. The time for incubation dependsprimarily on the temperature, being generally no greater than about 10hours to avoid an insensitive assay. Preferably, the incubation time isfrom about 0.5 to 3 hours, and more preferably 1.5-3 hours at roomtemperature to maximize binding of free VEGF₁₁₀₊ or VEGF to capturereagents. The duration of incubation may be longer if a proteaseinhibitor is added to prevent proteases in the biological fluid fromdegrading the VEGF.

At this stage, the pH of the incubation mixture will ordinarily be inthe range of about 4-9.5, preferably in the range of about 6-9, morepreferably about 7-8, and most preferably the pH of the assay (ELISA)diluent is pH 7.4. The pH of the incubation buffer is chosen to maintaina significant level of specific binding of the capture reagent to theVEGF₁₁₀₊ or VEGF being captured. Various buffers may be employed toachieve and maintain the desired pH during this step, including borate,phosphate, carbonate, Tris-HCl or Tris-phosphate, acetate, barbital, andthe like. The particular buffer employed is not critical to theinvention, but in individual assays one buffer may be preferred overanother.

Second Step

In the second step of the assay method herein, which is optional, thebiological sample is separated (preferably by washing) from theimmobilized capture reagent to remove uncaptured molecules. The solutionused for washing is generally a buffer (“washing buffer”) with a pHdetermined using the considerations and buffers described above for theincubation step, with a preferable pH range of about 6-9. The washingmay be done three or more times. The temperature of washing is generallyfrom refrigerator to moderate temperatures, with a constant temperaturemaintained during the assay period, typically from about 0-40° C., morepreferably about 4-30° C. For example, the wash buffer can be placed inice at 4° C. in a reservoir before the washing, and a plate washer canbe utilized for this step. A cross-linking agent or other suitable agentmay also be added at this stage to allow the now-bound VEGF₁₁₀₊ or VEGFto be covalently attached to the capture reagent if there is any concernthat the captured VEGF₁₁₀₊ or VEGF may dissociate to some extent in thesubsequent steps.

Third Step

In the next step, the immobilized capture reagent is contacted withdetectable antibodies, preferably at a temperature of about 20-40° C.,more preferably about 20-25° C., with the exact temperature and time forcontacting the two being dependent primarily on the detection meansemployed. For example, when strepatavidin-peroxidase and3,3′,5,5′-tetramethyl benzidine are used as the means for detection,e.g., in one embodiment, the contacting is carried out (e.g., about 1hour or more) to amplify the signal to the maximum. Preferably a molarexcess of an antibody with respect to the maximum concentration of freeVEGF₁₁₀₊ or VEGF expected (as described above) is added to the plateafter it is washed. This antibody is directly or indirectly detectable.While the detectable antibody may be a polyclonal or monoclonalantibody, e.g., in certain embodiments, it is a monoclonal antibody, inone embodiment murine, and in one embodiment MAb A4.6.1. Also, thedetectable antibody can be directly detectable, and in one embodimenthas a colorimetric label, and in another embodiment has a flurometriclabel. More preferably, the detectable antibody is biotinylated and thedetection means is avidin or streptavidin-peroxidase and3,3′,5,5′-tetramethyl benzidine. The readout of the detection means canbe fluorimetric or colorimetric. The affinity of the antibody must besufficiently high that small amounts of the free VEGF₁₁₀₊ or VEGF can bedetected, but not so high that it causes the VEGF₁₁₀₊ or VEGF to bepulled from the capture reagents.

Fourth Step

In the last step of the assay method, the level of free VEGF that is nowbound to the capture reagent is measured using a detection means for thedetectable antibody. If the biological sample is from a vascular,diabetic, or cancer patient, the measuring step preferably comprisescomparing the reaction that occurs as a result of the above three stepswith a standard curve to determine the level of VEGF₁₁₀₊ or VEGFcompared to a normal individual, or preferably comprises comparing thereaction that occurs as a result of the above three steps with ananother VEGF ELISA recognizing different isoforms or total VEGF todetermine the level of the types of VEGF when the ELISAs are compared,and optionally compared to a normal individual.

Antibody Production

Polyclonal antibodies to the VEGF generally are raised in animals bymultiple subcutaneous (sc) or intraperitoneal (ip) injections of theVEGF and an adjuvant. It may be useful to conjugate the VEGF or afragment containing the target amino acid sequence to a protein that isimmunogenic in the species to be immunized, e.g., keyhole limpethemocyanin, serum albumin, bovine thyroglobulin, or soybean trypsininhibitor using a bifunctional or derivatizing agent, for example,maleimidobenzoyl sulfosuccinimide ester (conjugation through cysteineresidues), N-hydroxysuccinimide (through lysine residues),glutaraldehyde, succinic anhydride, SOCl2, or R1N═C═NR, where R and R1are different alkyl groups.

The antibodies used as the coat or detectable antibodies may be obtainedfrom any convenient vertebrate source, such as murine, primate,lagomorpha, goat, rabbit, rat, chicken, bovine, ovine, equine, canine,feline, or porcine. Chimeric or humanized antibodies may also beemployed, as described, e.g., in U.S. Pat. No. 4,816,567; Morrison etal. Proc. Natl. Acad. Sci. USA 81:6851 (1984); Neuberger et al. Nature312: 604 (1984); Takeda et al. Nature 314:452 (1985); and WO 98/45331published Oct. 15, 1998, as well as in those additional references setforth above.

Animals may be immunized against the immunogenic conjugates orderivatives by combining 1 mg or 1 μg of conjugate (for rabbits or mice,respectively) with 3 volumes of Freund's complete adjuvant and injectingthe solution intradermally at multiple sites. One month later theanimals are boosted with ⅕ to 1/10 the original amount of conjugate inFreund's incomplete adjuvant by subcutaneous injection at multiplesites. 7 to 14 days later animals are bled and the serum is assayed foranti-VEGF titer. Animals are boosted until the titer plateaus.Preferably, the animal is boosted with the conjugate of VEGF, butconjugated to a different protein and/or through a differentcross-linking agent. Conjugates also can be made in recombinant cellculture as protein fusions. Also, aggregating agents such as alum areused to enhance the immune response. Methods for the production ofpolyclonal antibodies are described in numerous immunology textbooks,such as Davis et al. Microbiology, 3^(rd) Edition, (Harper & Row, NewYork, N.Y., 1980).

Monoclonal antibodies are prepared by recovering spleen cells fromimmunized animals and immortalizing the cells in conventional fashion,e.g. by fusion with myeloma cells or by Epstein-Barr virustransformation, and screening for clones expressing the desiredantibody. See, e.g., Kohler and Milstein Eur. J. Immunol. 6:511 (1976).Monoclonal antibodies, or the antigen-binding region of a monoclonalantibody, such as Fab or (Fab)₂ fragments, may alternatively be producedby recombinant methods.

Examples of suitable antibodies include those already utilized in knownRIAs for the protein in question, e.g., those antibodies directedagainst VEGF as described in the references given in the introductionherein.

In certain embodiments, an anti-VEGF antibody 5C3, which is obtainablefrom or produced by hybridoma deposited under ATCC number PTA-7737, isused, optionally with another anti-VEGF antibody, A4.6.1. The inventionalso provides an antibody that does not bind VEGF 1-110 and binds to thesame VEGF₁₁₀₊ epitope as the monoclonal antibody produced by hybridomacell line PTA-7737. A hybridoma 5C3.1.1 deposited under ATCC depositnumber PTA-7737 is provided.

Detection

The antibody added to the immobilized capture reagents will be eitherdirectly labeled, or detected indirectly by addition, after washing offof excess first antibody, of a molar excess of a second, labeledantibody directed against IgG of the animal species of the firstantibody. In the latter, indirect assay, labeled antisera against thefirst antibody are added to the sample so as to produce the labeledantibody in situ.

The label used for either the first or second antibody is any detectablefunctionality that does not interfere with the binding of free VEGF₁₁₀₊or VEGF to the antibody. Examples of suitable labels are those numerouslabels known for use in immunoassay, including moieties that may bedetected directly, such as fluorochrome, chemiluminscent, andradioactive labels, as well as moieties, such as enzymes, that must bereacted or derivatized to be detected. Examples of such labels includethe radioisotopes ³²P, ¹⁴C,¹²⁵I, ³H, ¹³¹I, fluorophores such as rareearth chelates or fluorescein and its derivatives, rhodamine and itsderivatives, dansyl, umbelliferone, luceriferases, e.g., fireflyluciferase and bacterial luciferase (U.S. Pat. No. 4,737,456),luciferin, 2,3-dihydrophthalazinediones, horseradish peroxidase (HRP),alkaline phosphatase, β-galactosidase, glucoamylase, lysozyme,saccharide oxidases, e.g., glucose oxidase, galactose oxidase, andglucose-6-phosphate dehydrogenase, heterocyclic oxidases such as uricaseand xanthine oxidase, coupled with an enzyme that employs hydrogenperoxide to oxidize a dye precursor such as HRP, lactoperoxidase, ormicroperoxidase, biotin/avidin, biotin/streptavidin,biotin/Streptavidin-β-galactosidase with MUG, spin labels, bacteriophagelabels, stable free radicals, and the like. As noted above, thefluorimetric detection is one example.

Conventional methods are available to bind these labels covalently toproteins or polypeptides. For instance, coupling agents such asdialdehydes, carbodiimides, dimaleimides, bis-imidates, bis-diazotizedbenzidine, and the like may be used to tag the antibodies with theabove-described fluorescent, chemiluminescent, and enzyme labels. See,for example, U.S. Pat. No. 3,940,475 (fluorimetry) and U.S. Pat. No.3,645,090 (enzymes); Hunter et al. Nature 144:945 (1962); David et al.Biochemistry 13:1014-1021 (1974); Pain et al. J. Immunol. Methods40:219-230 (1981); and Nygren J. Histochem. and Cytochem. 30:407-412(1982). In certain embodiments, labels herein are fluorescent toincrease amplification and sensitivity to 8 pg/ml, more preferablybiotin with streptavidin-β-galactosidase and MUG for amplifying thesignal. In certain embodiments, a colorimetric label is used, e.g.,where the detectable antibody is biotinylated and the detection means isavidin or streptavidin-peroxidase and 3,3′,5,5′-tetramethyl benzidine.

The conjugation of such label, including the enzymes, to the antibody isa standard manipulative procedure for one of ordinary skill inimmunoassay techniques. See, for example, O'Sullivan et al. “Methods forthe Preparation of Enzyme-antibody Conjugates for Use in EnzymeImmunoassay,” in Methods in Enzymology, ed. J. J. Langone and H. VanVunakis, Vol. 73 (Academic Press, New York, N.Y., 1981), pp. 147-166.

Following the addition of last labeled antibody, the amount of boundantibody is determined by removing excess unbound labeled antibodythrough washing and then measuring the amount of the attached labelusing a detection method appropriate to the label, and correlating themeasured amount with the amount of free VEGF₁₁₀₊ or VEGF in thebiological sample. For example, in the case of enzymes, the amount ofcolor developed and measured will be a direct measurement of the amountof VEGF₁₁₀₊ or VEGF present. Specifically, if HRP is the label, thecolor is detected using the substrate 3,3′,5,5′-tetramethyl benzidine at450 nm absorbance.

In one example, after an enzyme-labeled second antibody directed againstthe first unlabeled antibody is washed from the immobilized phase, coloror chemiluminiscence is developed and measured by incubating theimmobilized capture reagent with a substrate of the enzyme. Then theamount of free VEGF₁₁₀₊ or VEGF concentration is calculated by comparingwith the color or chemiluminescence generated by the standard VEGF runin parallel.

Kits

As a matter of convenience, the assay method of this invention can beprovided in the form of a kit. Such a kit is a packaged combinationincluding the basic elements of:

(a) capture reagent comprised of the monoclonal antibody against humanVEGF molecule, wherein the monoclonal antibody recognizes VEGF₁₁₀₊; and

(b) detection reagents comprised of detectable (labeled or unlabeled)antibodies that bind to the KDR and FLT1 receptor binding domains ofVEGF. These basic elements are defined hereinabove. In certainembodiment, the detection reagents comprise a detectable antibody(ies)that bind to epitope of VEGF1-110.

Preferably, the kit further comprises a solid support for the capturereagents, which may be provided as a separate element or on which thecapture reagents are already immobilized. Hence, the capture antibodiesin the kit may be immobilized on a solid support, or they may beimmobilized on such support that is included with the kit or providedseparately from the kit.

Preferably, the capture reagents are coated on a microtiter plate. Thedetection reagent may be labeled antibodies detected directly orunlabeled antibodies that are detected by labeled antibodies directedagainst the unlabeled antibodies raised in a different species. Wherethe label is an enzyme, the kit will ordinarily include substrates andcofactors required by the enzyme, and where the label is a fluorophore,a dye precursor that provides the detectable chromophore. Where thedetection reagent is unlabeled, the kit may further comprise a detectionmeans for the detectable antibodies, such as the labeled antibodiesdirected to the unlabeled antibodies, preferably in afluorimetric-detected format. Where the label is an enzyme, the kit willordinarily include substrates and cofactors required by the enzyme,where the label is a fluorophore, a dye precursor that provides thedetectable chromophore, and where the label is biotin, an avidin such asavidin, streptavidin, or streptavidin conjugated to HRP orβ-galactosidase with MUG.

In one specific embodiment, the capture reagent is monoclonal antibody,preferably rodent, more preferably murine or rat, still more preferablymurine, and most preferably MAb 5C3. Also in certain embodiments, thedetectable antibody is a biotinylated monoclonal antibody, themonoclonal antibody is rodent, more preferably murine or rat, still morepreferably murine, yet still more preferably MAb A4.6.1. In certainembodiments, the capture reagent is immobilized in this kit.

In certain embodiments, the kit can contain multiple ELISA forcomparison studies as described herein for detecting various forms ofVEGF and VEGF₁₁₀₊.

The kit also typically contains instructions for carrying out the assay,and/or VEGF as an antigen standard (e.g., purified VEGF, preferablyrecombinantly produced VEGF, and VEGF110), as well as other additivessuch as stabilizers, washing and incubation buffers, and the like.

Examples of standards for VEGF are recombinant human VEGF produced inmammalian cells available from Genentech, Inc., South San Francisco,Calif., and from those companies and processes described herein.

The components of the kit will be provided in predetermined ratios, withthe relative amounts of the various reagents suitably varied to providefor concentrations in solution of the reagents that substantiallymaximize the sensitivity of the assay. Particularly, the reagents may beprovided as dry powders, usually lyophilized, including excipients,which on dissolution will provide for a reagent solution having theappropriate concentration for combining with the sample to be tested.

Deposit of Materials

The following material has been deposited with the American Type CultureCollection, 10801 University Boulevard, Manassas, Va. 20110-2209, USA(ATCC):

5C3.1.1 was deposited with the ATCC under accession number PTA-7737deposited on Jul. 19, 2006.

Hybridoma ATCC Accession No. Deposit Date 5C3.1.1 PTA-7737 Jul. 19, 2006A4.6.1 HB10709 Mar. 29, 1991

The deposit was made under the provisions of the Budapest Treaty on theInternational Recognition of the Deposit of Microorganisms for thePurpose of Patent Procedure and the Regulations thereunder (BudapestTreaty). This assures maintenance of a viable culture of the deposit for30 years from the date of deposit. The deposits will be made availableby ATCC under the terms of the Budapest Treaty, and subject to anagreement between Genentech, Inc. and ATCC, which assures permanent andunrestricted availability of the progeny of the culture of the depositto the public upon issuance of the pertinent U.S. patent or upon layingopen to the public of any U.S. or foreign patent application, whichevercomes first, and assures availability of the progeny to one determinedby the U.S. Commissioner of Patents and Trademarks to be entitledthereto according to 35 USC §122 and the Commissioner's rules pursuantthereto (including 37 CFR §1.14 with particular reference to 886 OG638).

The assignee of the present application has agreed that if a culture ofthe materials on deposit should die or be lost or destroyed whencultivated under suitable conditions, the materials will be promptlyreplaced on notification with another of the same. Availability of thedeposited material is not to be construed as a license to practice theinvention in contravention of the rights granted under the authority ofany government in accordance with its patent laws.

The specification is considered to be sufficient to enable one skilledin the art to practice the invention. The invention is not to be limitedin scope by the construct deposited, since the deposited embodiment isintended as a single illustration of certain aspects of the inventionand any constructs that are functionally equivalent are within the scopeof the invention. The deposit of material herein does not constitute anadmission that the written description is inadequate to enable thepractice of any aspect of the invention, including the best morethereof, nor is it to be construed as limiting the scope of the claimsto the specific illustrations that it represents. Indeed, variousmodifications of the invention in addition to those shown and describedherein will become apparent to those skilled in the art from theforegoing description and fall within the scope of the appended claims.

It is understood that the examples and embodiments described herein arefor illustrative purposes only and that various modifications or changesin light thereof will be suggested to persons skilled in the art and areto be included within the spirit and purview of this application andscope of the appended claims. All publications, patents, and patentapplications cited herein are hereby incorporated by reference in theirentirety for all purposes.

EXAMPLES Example 1

Vascular endothelial growth factor (VEGF), which is expressed asdifferent isoforms due to alternative RNA splicing, is known to play akey role in tumor angiogenesis. We measured the concentrations ofVEGF₁₆₅ and total VEGF and evaluated the relative amount of VEGF₁₁₀,which is an active fragment generated by plasmin digestion of VEGF.ELISA A (VEGF165-206 ELISA) detects VEGF₁₆₅ and longer isoforms but notVEGF₁₂₁. ELISA B (VEGF110-206 ELISA) detects VEGF₁₆₅ and isoforms,VEGF₁₂₁ and VEGF₁₁₀. ELISA C (VEGF121-206 ELISA) detects VEGF₁₆₅ andlonger isoforms, VEGF₁₂₁ and VEGF fragments with molecular weight largerthan VEGF₁₁₀ but not VEGF₁₁₀ (referred to herein as “VEGF₁₁₀₊”).

Materials and Methods

Reagents and cells: Recombinant VEGF₁₆₅ (Genentech), VEGF₁₂₁ (PeproTech,Rocky Hill, N.J.), VEGF₈₋₁₀₉ (consisting of amino acids 8-109 ofVEGF₁₆₅) and truncated VEGF₁₂₁ (R&D Systems, Minneapolis, Minn.) wereproduced in E. coli. Truncated VEGF₁₂₁ has an intact N-terminus by massspectrometry but has a mass of 26 KDa, consistent with truncation ofapproximately nine amino acids from the carboxy-terminus according tothe manufacturer. It migrated between VEGF₁₁₀ and VEGF₁₂₁ when analyzedby SDS-PAGE under reducing conditions. VEGF₁₁₀ was prepared by plasmindigestion of VEGF₁₆₅ (Keyt B A, et al.,: The carboxyl-terminal domain(111-165) of vascular endothelial growth factor is critical for itsmitogenic potency. J Biol Chem. 271: 7788-7795 (1996)). The molecularweight measured by mass spectrometry was 25390, matching the theoreticalmass of 25389. The concentration was determined using bicinchorinic acidmethod (Pierce, Rockford, Ill.). Molecular weights used forconcentration calculation of VEGF₈₋₁₀₉, VEGF₁₂₁ and VEGF₁₆₅ were 23.8,28.9 and 38.2 KDa, respectively. Monoclonal anti-VEGF antibodies A4.6.1,3.5F8, 2E3 and 5C3 were generated by immunizing mice with VEGF₁₆₅produced in CHO cells (Kim K J, et al.,: The vascular endothelial growthfactor proteins: Identification of biologically relevant regions byneutralizing monoclonal antibodies. Growth Factors 7: 53-64 (1992)).Breast cell lines SK-BR-3, BT-474, T-47D and MCF-7 as well as ovariancell lines ES-2, OVCAR-3 and SK-OV-3 (American Type Culture Collection,Rockville, Md.) were grown in RPMI, 2 mM L-glutamine and 10% FBS (except20% for OVCAR-3) in a humidified 5% CO₂ incubator at 37° C.

Purification of VEGF in conditioned media of A673 cells: A673 cells(American Type Culture Collection,) were grown in 50:50 F12/DMEM, 2 mML-glutamine and 5% FBS to 60% confluency and then in serum free medium(Genentech) till confluency. VEGF was purified from the supernatantsusing an A4.6.1-Sepharose column that was prepared with CNBr activatedSepharose (Amersham Biosciences, Piscataway, N.J.). The column eluateand recombinant VEGF controls (0.2 μg per lane) were run on a 18%Tris-Glycine gels (Invitrogen, Carlsbad, Calif.) under reducingconditions and were blotted unto nitrocellulose. The blot was blockedwith 0.5M Tris-HCl, pH 7.5, 1.5M NaCl, 50 mM EDTA, 0.5% Trition100containing 3% bovine serum albumin and probed with 200 ng/ml of 3.5F8 orA4.6.1 followed by 2 ng/ml of goat anti-mouse Fc-HRP (JacksonImmunoResearch). Signals were developed using SuperSignal West Dura(Pierce) and recorded on X-ray film.

VEGF ELISAs for Measuring VEGF Concentrations

ELISA A (VEGF165-206 ELISA). Unless otherwise mentioned, a fluorometricELISA A was used for measuring VEGF in samples. The fluorimetric ELISA Aused 3.5F8 for coat and biotinylated A4.6.1 followed by streptavidin-βgalactosidase for detection and 4-methylumbelliferyl-β-D galactoside asthe substrate (Rodriguez C R, et al.,: A sensitive fluorometricenzyme-linked immunosorbent assay that measures vascular endothelialgrowth factor165 in human plasma. J Immunol Methods 219: 45-55 (1998)).The VEGF₁₆₅ standards were 1-128 pg/mL, or 0.026-3.35 pM. Thecolorimetric ELISA A used 3.5F8 for coat and biotinylated A4.6.1 fordetection, following the protocol used for the ELISA C described below.The VEGF₁₆₅ standards were 1.6-200 pg/mL.

ELISA B (VEGF110-206 ELISA) (previously named VEGF121-206 ELISA, KonecnyG E, et al.,: Association between HER-2/neu and Vascular EndothelialGrowth Factor Expression Predicts Clinical Outcome in Primary BreastCancer Patients. Clinical Cancer Research, 10: 1706-1716 (2004)):MaxiSorp 96-well microwell plates were coated with 0.5 μg/ml antibodyA4.6.1 in 50 mM carbonate buffer, pH 9.6 at 100 μl/well at 4° C.overnight. Plates were washed after this step and between the subsequentroom temperature incubation steps with PBS, pH 7.4, containing 0.05%polysorbate 20. Plates were blocked with 0.5% bovine serum albumin, 10ppm Proclin™ 300 (Supelco, Bellefonte, Pa.) in PBS (150 μl/well) for 1h. VEGF standards (1.56-200 pg/ml VEGF₁₆₅ or 0.0409-5.24 pM VEGF intwofold serial dilution) and serially diluted samples (minimum 1:10dilution) in twofold or threefold serial dilution in PBS, pH 7.4,containing 0.5% bovine serum albumin, 0.05% polysorbate 20, 5 mM EDTA,0.25% CHAPS, 0.2% bovine γ-globulins (Sigma, St. Louis, Mo.) and 0.35 MNaCl (sample buffer) were added to the plates (100 μl/well) andincubated for 2 h. Bound VEGF was detected by incubating biotinylated2E3 (or another antibody that binds to a receptor binding domain ofVEGF) on the plates for 1 h followed by streptavidin-HRP (Amersham,Copenhagen, Denmark) for 30 min, biotinyl-tyramide (ELAST ELISAamplification System, Perkin Elmer Life Sciences Inc., MA) for 15 minand streptavidin-HRP for 30 min. The substrate TMB(3,3′,5,5′-tetramethyl benzidine) (Kirkegaard & Perry Laboratories) wasadded and the reaction was stopped by adding 1 M phosphoric acid.Absorbance was read at 450 nm on a Titertek stacker reader (ICN, CostaMesa, Calif.). The titration curves were fit using a four-parameterregression curve-fitting program (KaleidaGraph, Synergy software,Reading, Pa.). Data points which fell in the range of the standard curvewere used for calculating the putative VEGF concentrations in thesamples. The recovery of 1.56-200 pg/ml VEGF₁₆₅ in 10% human EDTA plasma(Golden West Biologicals Inc., Temecula, Calif.) was 92-120% aftersubtracting the putative 2.1 pg/ml endogenous VEGF in the 10% plasmaused for this study.

ELISA C (VEGF121-206 ELISA): Microwell plates were coated with 1 μg/mlanti-VEGF 5C3 antibody and blocked as described above. VEGF standards(4.00-512 pg/ml VEGF₁₆₅ or 0.105-13.4 pM VEGF in 2-fold serial dilution)and serially diluted samples in sample buffer were added to the plates.The plates were incubated for 2 h. Bound VEGF was detected by addingbiotinylated A4.6.1 followed by streptavidin-HRP and TMB as thesubstrate. Plates were read and data were analyzed as described above.The recovery of 4.00-512 pg/ml VEGF₁₆₅ in 10% plasma was 77-101% aftersubtracting 1.6 pg/ml putative endogenous VEGF in the 10% plasma usedfor this study.

Results and Discussion

VEGF ELISAs: The previously described ELISA A uses 3.5F8 for coat andbiotinylated A4.6.1 for detection (Rodriguez C R, et al.,: A sensitivefluorometric enzyme-linked immunosorbent assay that measures vascularendothelial growth factor165 in human plasma. J Immunol Methods 219:45-55, 1998). It detects VEGF165 (VEGF₁₆₅) but not VEGF121(1)(VEGF₁₂₁(1)), which is from R&D systems and missing approximately 9amino acids from the carboxy-terminus, and VEGF121(2) (VEGF₁₂₁(2)),which is from PeproTech (FIG. 1A). 3.5F8 binds VEGF₁₆₅ but not VEGF₁₂₁by BIAcore. A4.6.1 binds to the receptor binding domain (Kim K J, etal.,: The vascular endothelial growth factor proteins: Identification ofbiologically relevant regions by neutralizing monoclonal antibodies.Growth Factors 7: 53-64, 1992) that is present in all isoforms and inVEGF₁₁₀. 3.5F8 likely binds near amino acids 116 and 118, which are notpresent in VEGF₁₂₁. 5C3 likely binds near amino acids 111-113, which arenot present in VEGF₁₁₀ (FIG. 3). ELISA A can likely detect VEGF isoformswhich contained VEGF₁₆₅ sequences including VEGF₁₈₃, VEGF₁₈₉ and VEGF₂₀₆(see, e.g., Stimpfl M, et al.,: Vascular Endothelial growth factorsplice variants and their prognostic value in breast and ovarian cancer.Clinical Cancer Research 8: 2253-2259, 2002). ELISA B (previously namedVEGF121-206 ELISA, Konecny G E, et al., Association between HER-2/neuand Vascular Endothelial Growth Factor Expression Predicts ClinicalOutcome in Primary Breast Cancer Patients. Clinical Cancer Research, 10:1706-1716, 2004) uses A4.6.1 for coat and biotinylated 2E3 fordetection. A4.6.1 and 2E3 bind to the receptor binding domain that ispresent in all three molecules. See, e.g., Kim K J, et al. The vascularendothelial growth factor proteins: Identification of biologicallyrelevant regions by neutralizing monoclonal antibodies. Growth Factors7:53-64 (1992); and, Muller Y A, et al. Vascular endothelial growthfactor: Crystal structure and functional mapping of the kinase domainreceptor binding site. Proc Natl Acad Sci USA 94:7192-7197 (1997). Otherantibodies that bind in these regions can also be used. This ELISAdetects VEGF₁₆₅, VEGF₁₂₁, truncated VEGF₁₂₁ (missing approximately 9amino acids from the carboxy-terminus), VEGF₁₁₀ and VEGF₈₋₁₀₉ equallywell (FIG. 1B). This ELISA can detect total VEGF, including fragmentslarger than VEGF₁₁₀ generated by matrix metalloproteinase digestion.ELISA C, described herein, which uses 5C3 for coat and biotinylatedA4.6.1 for detection, detects VEGF₁₆₅, VEGF₁₂₁, and truncated VEGF₁₂₁equally well but does not detect VEGF₁₁₀ or VEGF₈₋₁₀₉ (FIG. 1, C). 5C3binds VEGF₁₂₁ but not VEGF₈₋₁₀₉ by BIAcore. This ELISA can detect allthe VEGF molecules detected by the VEGF₁₁₀₋₂₀₆ except VEGF₁₁₀ andsmaller fragments.

The sensitivities of ELISA A, ELISA B and ELISA C were 10, 16 and 40pg/ml VEGF₁₆₅ (or 0.26, 0.41 and 1.05 pM for different VEGF isoforms andfragments) for VEGF in samples using a minimum 1:10 dilution,respectively. ELISA B and ELISA C were reproducible (Table 1 & 2). ELISAB and ELISA C were specific to VEGF (VEGF-A). VEGF-B, VEGF-C and VEGF-Dat concentrations up to 50 ng/ml only gave background signals.Insulin-like growth factor 1, growth hormone, recombinant nerve growthfactor, tumor necrosis factor (Genentech), platelet-derived growthfactor AB, placenta growth factor, transforming growth factor β1 (R&DSystems) (up to 200 ng/ml) only gave background signals. Heparin (LeoLaboratories, Bucks, UK and Dublin, Ireland) (up to 100 U/ml) did nothave a significant effect on the assay.

TABLE 1 ELISA B (VEGF₁₁₀₋₂₀₆ ELISA): The standard range was 1.56-200pg/ml VEGF₁₆₅ (0.0409-5.24 pM VEGF) in buffer. The OD ratio of 1.56pg/ml standard relative to the blank was 1.37 ± 0.11. CV is coefficientof variation. Mean Inter Intra Control^(a) (pg/ml) % CV % CV Low 3.0717.7 13.5 Middle 38.0 9.50 6.54 High 127 9.11 6.95 ^(a)The middle andhigh controls were made by spiking recombinant VEGF₁₆₅ into human EDTAplasma. The low control was made by spiking VEGF₁₆₅ into 70% plasmasince plasma contained endogenous VEGF. Controls were diluted 1:10 andassayed in duplicate in 34 independent assays.

TABLE 2 ELISA C (VEGF₁₂₁₋₂₀₆ ELISA). The standard range was 4.00-512pg/ml VEGF₁₆₅ (0.105-13.4 pM VEGF). The OD ratio of 4 pg/ml standardrelative to the blank was 2.72 ± 0.37. CV is coefficient of variation.Mean Inter Intra Control^(a) (pg/ml) % CV % CV Low 3.28 20.6 8.35 Middle11.7 6.56 2.39 High 56.5 2.57 1.37 ^(a)The controls were made by spikingrecombinant VEGF₁₆₅ into human EDTA plasma. They were diluted 1:10 andassayed in duplicate in 15 independent assays.

VEGF in conditioned media of cell lines: Conditioned media from sixstable CHO clones transfected with VEGF₁₆₅ cDNA (Meng et al., 2000) weremeasured by the three ELISAs, which used non-glycosylated VEGF producedin E. coli as standard. Glycosylated recombinant VEGF₁₆₅ in conditionedmedia from six stable CHO clones gave very similar concentrations in thethree ELISAs. Concentrations measured by ELISA B were 28, 63, 64, 43,3.8 and 3.2 nM, respectively. Ratios of VEGF concentrations measured byELISA A and ELISA C compared to those by ELISA B were 0.90±0.08 and1.08±0.10, respectively. Therefore, the three ELISAs quantitatedglycosylated VEGF equally well and there was little proteolysis ofVEGF₁₆₅ under the culture conditions.

VEGF concentrations in A673 cell conditioned medium measured by ELISA A,ELISA B and ELISA C were 0.15, 0.29 and 0.24 nM VEGF, respectively. Theconcentration measured by ELISA A was lower, indicating VEGF₁₂₁ waspresent. When VEGF was purified from conditioned medium using an A4.6.1affinity column and analyzed by protein blotting, two bands, likelyglycosylated and non-glycosylated VEGF₁₆₅ were detected by 3.5F8. Thelower band had the same mobility as the purified VEGF₁₆₅ produced in E.coli (FIG. 2, left). N-glycanase treatment converted the upper band tothe lower band. Two additional lower molecular weight bands, likelyglycosylated (partially overlapping with the putative non-glycosylatedVEGF₁₆₅ band) and non-glycosylated VEGF₁₂₁ were detected by A4.6.1 (FIG.2, right). The lower band had the same mobility as the purified VEGF₁₂₁produced in E. coli and N-glycanase treatment converted the upper bandto the lower band.

VEGF concentrations in conditioned media from breast cell lines SK-BR-3,BT-474, T-47D and MCF-7 measured by ELISA B were 3.6, 16, 13, and 13 pM,respectively. Ratios of VEGF concentrations measured by ELISA A to thoseby ELISA B were 0.49, 0.42, 0.43 and 0.38 (or 49%, 42%, 43% or 38%),respectively, in agreement with 43, 35, 40 and 41% of VEGF₁₆₅ expressionin these respective cell lines (Stimpfl M, et al.,: Vascular Endothelialgrowth factor splice variants and their prognostic value in breast andovarian cancer. Clinical Cancer Research 8: 2253-2259, 2002). Ratios ofVEGF concentrations measured by ELISA C to those by ELISA B were 1.1-1.2for these cell lines, indicating that little VEGF₁₁₀ was present. VEGFconcentrations in conditioned media from ovarian cell lines ES-2,OVCAR-3 and SK-OV-3 measured by ELISA B were 32, 11 and 20 pM,respectively. Ratios of VEGF concentrations measured by ELISA A to thoseby ELISA B were 0.24, 0.20, and 0.32 (or 24%, 20% and 32%),respectively, compared to 38, 42 and 24% of VEGF₁₆₅ expression in theserespective cell lines (Stimpfl et al., supra). Ratios of VEGFconcentrations measured by ELISA C to those by ELISA B were 0.64-0.79for these cell lines, indicating VEGF₁₁₀ (or smaller fragments) may bepresent.

We claim:
 1. An immunoassay kit for selectively detecting VEGF₁₁₀₊ formsin a biological sample, the forms including VEGF₁₂₁, the kit comprising:(a) as capture reagent, a monoclonal antibody against human VEGF,wherein the monoclonal antibody binds specifically to the residuesgreater than 110 of human VEGF and binds VEGF₁₂₁; and (b) as detectionreagent, a detectable antibody that binds to the KDR and/or FLT1receptor binding domains of VEGF or that binds to an epitope in VEGF1-110.
 2. The kit of claim 1, further comprising a solid support for thecapture reagent.
 3. The kit of claim 2, wherein the capture reagent isimmobilized on the solid support.
 4. The kit of claim 3, wherein thecapture reagent is coated on a microtiter plate.
 5. The kit of claim 1,further comprising a detection means for the detectable antibody.
 6. Thekit of claim 5, wherein the detection means is colorimetric.
 7. The kitof claim 1, wherein the detectable antibody is amplified by afluorimetric reagent.
 8. The kit of claim 1, wherein the detectableantibody is biotinylated and the detection means is avidin orstreptavidin-peroxidase and 3,3′,5,5′-tetramethyl benzidine.
 9. The kitof claim 1, wherein the detectable antibody is a monoclonal antibody.10. The kit of claim 9, wherein the detectable antibody is a murinemonoclonal antibody.
 11. The kit of claim 1, further comprising purifiedVEGF as an antigen standard.
 12. The kit of claim 1, wherein the capturereagent recognizes the same epitope as antibody 5C3.
 13. The kit ofclaim 12, wherein the capture reagent is a 5C3 monoclonal antibody. 14.The kit of claim 1, wherein the capture reagent is murine monoclonalantibody MAb 5C3 and the detectable antibody is MAb A4.6.1.
 15. The kitof claim 1, wherein said kit further selectively detects the form,VEGF₁₆₅.